The plasmid mediated spread of metallo-beta-lactamase poses a growing threat against the ability to combat antibiotic-resistant bacteria. This newly emerging enzyme has evolved to catalyze the hydrolysis of most beta- lactam antibiotics in clinical use. Understanding how this beta-lactamase works and how it can be inhibited is of significant economic and humanitarian interest. Found at the active site are two co-catalytic zinc ions, the functions of which are still poorly understood. This proposal employs site-directed mutagenesis and a zinc chelator to study metallo-beta- lactamases that can bind either one or two zinc ions selectively. Pre-steady- state and steady-state kinetics, mutagenesis spectroscopy, and inhibitor studies will be used to identify modes of inhibitor binding, the role of the protein environment and how the evolution of a second zinc binding site has resulted in a particularly effective and dangerous catalyst. Information about the catalytic mechanism and inhibitor binding will contribute to our understanding of dinuclear hydrolases and to the design of needed antibacterial agents combating the emerging threat of antibiotic-resistant bacteria.